Long-term preservation of cones and improvement in visual function following gene therapy in a mouse model of leber congenital amaurosis caused by guanylate cyclase-1 deficiency.

Leber congenital amaurosis (LCA) is a severe retinal dystrophy manifesting from early infancy as poor vision or blindness. Loss-of-function mutations in GUCY2D cause LCA1 and are one of the most common causes of LCA, accounting for 20% of all cases. Human GUCY2D and mouse Gucy2e genes encode guanylate cyclase-1 (GC1), which is responsible for restoring the dark state in photoreceptors after light exposure. The Gucy2e(-/-) mouse shows partially diminished rod function, but an absence of cone function before degeneration. Although the cones appear morphologically normal, they exhibit mislocalization of proteins involved in phototransduction. In this study we tested the efficacy of an rAAV2/8 vector containing the human rhodopsin kinase promoter and the human GUCY2D gene. Following subretinal delivery of the vector in Gucy2e(-/-) mice, GC1 protein was detected in the rod and cone outer segments, and in transduced areas of retina cone transducin was appropriately localized to cone outer segments. Moreover, we observed a dose-dependent restoration of rod and cone function and an improvement in visual behavior of the treated mice. Most importantly, cone preservation was observed in transduced areas up to 6 months post injection. To date, this is the most effective rescue of the Gucy2e(-/-) mouse model of LCA and we propose that a vector, similar to the one used in this study, could be suitable for use in a clinical trial of gene therapy for LCA1.

Dominant cone-rod dystrophy: a mouse model generated by gene targeting of the GCAP1/Guca1a gene.

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.

Neuroprotective gene therapy for the treatment of inherited retinal degeneration.

Inherited retinal degeneration (IRD) affects around 1/3000 of the population in Europe and the United States. It is a diverse group of conditions that results from mutations in any one of over 100 different genes. Many of the genes have now been identified and their functions elucidated, providing a major impetus to develop gene-based treatments. Whilst gene replacement and gene silencing strategies offer prospects for the treatment of specific inherited retinal disorders, other disorders may be less amenable to these corrective approaches. These conditions include, in particular, those associated with abnormal retinal development and those in which retinal degeneration is advanced at birth. Furthermore, the development of individualized corrective gene therapy strategies for patients with disorders due to very rare mutations may be unfeasible. However, generic gene therapy strategies that aim not to correct the gene defect but to ameliorate its consequences offer the possibility of therapies that are widely applicable across a range of conditions. One potential strategy in these cases is to halt or delay the process of cell death, so that useful visual function can be maintained throughout the lifetime of an affected individual. It has been shown in variety of experimental models over the last three decades, that neurotrophic factors have the potential to delay neuronal apoptosis. Neurotrophic factors are small proteins which have relatively short half lives and a requirement for repeated administration has limited their clinical application. Since these proteins do not ordinarily cross the blood-brain barrier, previous approaches have relied upon intrathecal infusion pumps or similar complex devices to sustain elevated neurotrophin levels within the central nervous system (CNS). However, sustained delivery through viral vector mediated expression of genes encoding neurotrophic factors may circumvent the potential side effects of repeated administration. In this review we shall explore some of the concepts of neurotrophic gene therapy and how this might be applicable to preserving vision in inherited retinal degenerations.

CNTF gene transfer protects ganglion cells in rat retinae undergoing focal injury and branch vessel occlusion.

Ciliary neurotrophic factor (CNTF) has been shown to protect ganglion cells in a variety of acute ischaemia models. Here we assess the efficacy of local CNTF gene transfer in protecting retinal ganglion cells when there is focal ischaemia combined with interruption of axoplasmic flow. This dual injury may be more representative of the pathological mechanisms operating in acute retinal diseases, such as vascular events acting at the optic nerve head. Fourteen rats received an intravitreal injection of an adeno-associated viral (AAV) vector expressing a secretable form of CNTF into the right eye and a control vector into the left eye. Three weeks later, each rat underwent a symmetrical small vertical 2mm standardised retinal crush injury approximately 2mm temporal to the optic disc. The injury also occluded the temporal retinal arteriole so that the axon crush was combined with an acute retinal infarction visible on fundoscopy. Changes in the damaged sector were compared histologically four weeks after injury and ganglion cell survival was estimated by comparing cell counts on retinal flat-mounts immunostained with RT-97 antibody. This mode of injury led to a profound loss of both the inner nuclear and ganglion cell layers, but was limited to the lesioned sector. In AAV.CNTF-treated eyes approximately 12% of ganglion cells survived compared with approximately 2% in control eyes (p=0.01). The scotopic electroretinogram (ERG), however, was reduced to about 50% in AAV.CNTF-treated eyes, both before and after injury. We therefore show that CNTF gene transfer confers neuroprotection to ganglion cells undergoing an acute ischaemic injury combined with interruption of axoplasmic flow. This approach may be relevant to optic nerve trauma and a variety of retinal vascular diseases that lead to swelling of the optic nerve head, provided CNTF can be delivered in a way that does not significantly suppress retinal function.

In contrast to AAV-mediated Cntf expression, AAV-mediated Gdnf expression enhances gene replacement therapy in rodent models of retinal degeneration.

While AAV- and lentivirus-mediated gene replacement therapy can produce structural and functional improvements in various animal models of inherited retinal degeneration, this approach often has very limited effects on the rate of photoreceptor cell loss. Neurotrophic factors such as ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) have been shown to prolong photoreceptor survival in rodent models of retinal degeneration, but AAV-mediated Cntf expression also results in suppression of electrophysiological responses from the retina. In this study using mice, we show that while the deleterious effects mediated by CNTF are dose-dependent, administering a dose of CNTF that does not adversely affect retinal function precludes its ability to delay photoreceptor cell death. In evaluating GDNF as a neuroprotective agent, we show that AAV-mediated Gdnf expression does not produce adverse effects similar to those of CNTF. In addition, we demonstrate the ability of AAV-mediated delivery of Gdnf to slow cell death in two rodent models of retinitis pigmentosa and to enhance retinal function in combination with the relevant gene replacement therapy. These data show for the first time that a combination of these approaches can provide enhanced rescue over gene replacement or growth factor therapy alone.

Gene replacement therapy rescues photoreceptor degeneration in a murine model of Leber congenital amaurosis lacking RPGRIP.

PURPOSE: Retinitis pigmentosa GTPase regulator (RPGR) is a photoreceptor protein anchored in the connecting cilia by an RPGR-interacting protein (RPGRIP). Loss of RPGRIP causes Leber congenital amaurosis (LCA), a severe form of photoreceptor degeneration. The current study was an investigation of whether somatic gene replacement could rescue degenerating photoreceptors in a murine model of LCA due to a defect in RPGRIP. METHODS: An RPGRIP expression cassette, driven by a mouse opsin promoter, was packaged into recombinant adeno-associated virus (AAV). The AAV vector was delivered into the right eyes of RPGRIP(-/-) mice by a single subretinal injection into the superior hemisphere. The left eyes received a saline injection as a control. Full-field electroretinograms (ERGs) were recorded from both eyes at 2, 3, 4, and 5 months after injection. After the final follow-up, retinas were analyzed by immunostaining or by light and electron microscopy. RESULTS: Delivery of the AAV vector led to RPGRIP expression and restoration of normal RPGR localization at the connecting cilia. Photoreceptor preservation was evident by a thicker cell layer and well-developed outer segments in the treated eyes. Rescue was more pronounced in the superior hemisphere coincident with the site of delivery. Functional preservation was demonstrated by ERG. CONCLUSIONS: AAV-mediated RPGRIP gene replacement preserves photoreceptor structure and function in a mouse model of LCA, despite ongoing cell loss at the time of intervention. These results indicate that gene replacement therapy may be effective in patients with LCA due to a defect in RPGRIP and suggest that further preclinical development of gene therapy for this disorder is warranted.

Local administration of an adeno-associated viral vector expressing IL-10 reduces monocyte infiltration and subsequent photoreceptor damage during experimental autoimmune uveitis.

Autoimmune posterior uveitis is a chronic, potentially blinding inflammatory disease of the eye. It is commonly treated with immunosuppressive drugs that have adverse long-term effects. Advances in gene transfer techniques have enabled long-term, stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. Here we report for the first time that subretinal injection of rAAV-2 encoding murine IL-10 into the retina of C57BL/6 mice significantly decreases the median experimental autoimmune uveitis (EAU) disease severity. This protection is shown to be due to a decrease in the number and activation status of infiltrating monocytes during EAU, as determined by costimulatory molecule expression and nitrotyrosine detection. No differences within splenocyte proliferative responses or serum antibody levels were detected, emphasizing the potential of gene therapy strategies in ameliorating autoimmune responses in local microenvironments without unwanted systemic effects.